NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of Delta(5)-3-ketosteroid isomerase
NMR evidence for the participation of a low-barrier hydrogen bond in the mechanism of Delta(5)-3-ketosteroid isomerase
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DOI：
10.1073/pnas.93.16.8220
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发表时间：
1996-08-06
影响因子：
11.1
通讯作者：
Mildvan, AS
中科院分区：
文献类型：
--
作者：
Zhao, QJ;Abeygunawardana, C;Mildvan, AS
δ（5）-3-酮类固醇异构酶（EC 5.3.3.1）促进烯丙基重排，其涉及经由二烯醇中间体的分子内质子转移。这种酶将催化速率提高了10倍（10）。两个残基，Tyr-14，使类固醇3-羰基极化并促进烯醇化的一般酸，和Asp-38，提取并将4 β-质子转移到6 β-位置的一般碱，分别对速率增加贡献10（4.7）和10（5.6）。一个主要的机理之谜是催化基团和它们的靶点的pK（a）值之间的巨大差异。当二烯醇中间体的类似物与异构酶结合时，质子NMR检测到在18.15 ppm处靠近芳香族质子的高度去屏蔽共振，并且对氚的优先性是氘的3倍（分馏因子phi = 0.34），与Tyr-14形成短、强（低势垒）氢键一致。该氢键的强度估计为至少7.1 kcal/mol。该键相对难以接近本体溶剂并且对pH不敏感。Tyr-14与中间体的低势垒氢键结合，以及先前证明的Asp-38对质子转移的隧穿贡献，为该异构酶的高催化能力提供了合理的定量解释。
Delta(5)-3-Ketosteroid isomerase (EC 5.3.3.1) promotes an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate. This enzyme enhances the catalytic rate by a factor of 10(10). Two residues, Tyr-14, the general acid that polarizes the steroid 3-carbonyl group and facilitates enolization, and Asp-38 the general base that abstracts and transfers the 4 beta-proton to the 6 beta-position, contribute 10(4.7) and 10(5.6) to the rate increase, respectively. A major mechanistic enigma is the huge disparity between the pK(a) values of the catalytic groups and their targets, Upon binding of an analog of the dienolate intermediate to isomerase, proton NMR detects a highly deshielded resonance at 18.15 ppm in proximity to aromatic protons, and with a 3-fold preference for protium over deuterium (fractionation factor phi = 0.34), consistent with formation of a short, strong (low-barrier) hydrogen bond to Tyr-14, The strength of this hydrogen bond is estimated to be at Least 7.1 kcal/mol. This bond is relatively inaccessible to bulk solvent and is pH insensitive. Low-barrier hydrogen bonding of Tyr-14 to the intermediate, in conjunction with the previously demonstrated tunneling contribution to the proton transfer by Asp-38, provide a plausible and quantitative explanation for the high catalytic power of this isomerase.